Human pluripotent stem cells (hPSCs) are now widely used to provide a theoretically endless and also large supply of human cardiomyocytes (Kehat et al. J Clin Invest 108, 407-414 (2001); Takahashi et al. Cell 131, 861-872 (2007); Zhang et al., Circ Res 104, e30-41 (2009)). Human cardiomyocytes have been derived from human embryonic stem cells (hESCs) (Thomson et al. Science 282, 1145-1147 (1998)) and induced pluripotent stem cells (hIPSCs) (Takahashi et al., Cell 131, 861-872 (2007)) and have a demonstrated use for multiple purposes including developmental models (Lian et al. Stem Cells 2012 (2012)), drug efficacy and/or safety screening (Schaaf et al. PLoS ONE 6, 20 (2011)), hypertrophy modelling and regenerative applications. Additionally, with recent advances in hIPSC technology, cardiomyocytes exhibiting heritable genetic disease phenotypes can be generated in vitro (Carvajal-Vergara, X. et al. Nature 465, 808-812 (2010); Itzhaki et al., Nature 471, 225-229 (2011); Malan et al. Circ Res (2011); Moretti et al., N Engl J Med 363, 1397-1409 (2010); Yazawa et al. Nature 471, 230-234 (2010)).
It is now widely accepted that the low density 2D culture of biopsy-derived human cardiomyocytes leads to rapid changes in cardiomyocyte phenotype and morphology (Bird et al. Cardiovasc Res 58, 423-434 (2003)) and making it difficult to extrapolate results to the in vivo situation. In order to obtain a cardiomyocyte phenotype more representative of in vivo conditions, cardiac tissue engineering has been used (Eschenhagen et al. FASEB J 11, 683-694 (1997); Zimmermann et al. Biotechnol Bioeng 68, 106-114 (2000), Zimmermann et al. Circ Res 90, 223-230 (2002); Tulloch et al. Circ Res 109, 47-59 (2011); Tiburcy et al. Circ Res 109, 1105-1114 (2011); Eschenhagen et al. Am J Physiol Heart Circ Physiol 303, 11 (2012)) to generate constructs with similar properties to the native heart tissue.
The current ideology of tissue engineering is to generate/isolate the required cell type(s), and seed them into an engineered environment to promote their differentiation and generate in vivo-like tissues. Tissue engineering may therefore be considered as an inefficient process for two reasons, 1) disassociation of a tissue/differentiation culture destroys the extracellular environment thus destroying developmental information (eg. cell-cell interconnectivity, geometric cell positioning, cell-ECM connectivity), this necessitates very large increases in extracellular matrix (ECM) production in order to re-build the environment (Hudson et al. Tissue Eng Part A 17, 2279-2289 (2011)), and 2) the disassociation process is variable between hPSC lines and can lead to considerable cell death.
Other protocols reported in the literature may require modification of the protocol to enable similar cardiomyocyte efficiencies in multiple hPSC lines. However, the inventor's results demonstrate that changes in differentiation protocol may greatly affect the cardiomyocyte phenotype (e.g. it is shown that dorsomorphin may greatly affect the bioengineered heart muscle (BHM)). This may lead to changes in tissue engineered myocardial properties which may mask the effects of different experimental conditions or genetic disease models, therefore care must be taken when using different protocols in different lines.
Some recently published protocols may enable the same protocol to be used for multiple lines, they also produce cardiomyocytes with very high purity. However, pure cardiomyocytes do not facilitate the formation of functional tissue engineered myocardium and both cardiomyocytes and stromal cells are required for the formation of functional tissue engineered myocardium (Naito et al. Circulation 114, 172-78 (2006), Hudson et al. Tissue Eng Part A 17, 2279-2289 (2011)).
Hence, there is a need in the art for methods for producing bioengineered human myocardium, which are capable of overcoming the above disadvantages.
The development of a robust differentiation protocol is a very important step allowing the consistent production of BHM. In this study n>140 BHM in >18 independent experiments were produced and every one exhibited spontaneous beating activity. Additionally, the protocol enables to produce BHM from multiple hPSC lines using the same protocol. In addition, all disassociation steps could be eliminated and hPSCs were differentiated directly into bioengineered myocardium, thus retaining the developmental memory of the tissue, prevent any tissue recreation response and provide a more accurate in vitro model of human myocardial development.